Plants and animals have yielded a number of chemical molecules having useful biological activity (e.g., anti-tumor activity). Particularly rich sources of biologically active chemicals are marine organisms, which comprise over half a million species. Marine organisms have been found to produce a variety of metabolic often having unprecedented chemical structures.
In recent years, an increasing number of natural products extracted from marine organisms have been reported to exhibit a variety of biological activities such as antimicrobial, antiviral, antifungal and anticancer activities. These include peptides, polyethers, alkaloids, prostanoids, and the like. Such compounds have been obtained from sponges, octocorals, algae, tunicates, nuclibranches, bryozoans and marine bacteria.
In particular, a number of anti-tumor and anti-fungal compounds have been extracted from marine life. For example, U.S. Pat. No. 4,729,996 discloses anti-tumor imidazole ring compounds isolated from the marine sponges Teichaxinella morchella and Ptioocaulis walpersi. U.S. Pat. No. 4,808,590 discloses nitrogen containing cyclic compounds isolated having antiviral, anti-tumor, and antifungal properties, isolated from the marine sponge Theoneloa sp. Similarly, U.S. Pat. No. 4,866,084 discloses bisindole alkaloids extracted from the marine sponge Spongosorites ruetzleri useful in treating certain classes of tumors, while U.S. Pat. No. 4,970,226 discloses bis-indole imidazole alkaloids and derivatives isolated from the marine sponge Spongosorites sp. which exhibit useful anti-tumor and antimicrobial properties.
Marine sponges, in particular, have proven to be a rich resource for biologically active compounds (see, e.g., Scheuer, P. J. (ed.) (1978-1983) Marine Natural Products, Chemical and Biological Perspectives Vol. I-V, Academic Press, New York; Faulkner (1977) Tetrahedron, 33: 1421; Faulkner (1984) Nat, Prod. Rep. 1: 551; Faulkner (1986) Nat, Prod. Rep. 3: 1; Faulkner (1987) Nat, Prod. Rep. 4: 539; Faulkner (1988) Nat, Prod. Rep. 5: 613; Faulkner (1990) Nat, Prod. Rep. 7: 269; Faulkner (1991) Nat, Prod. Rep. 7: 269; Faulkner (1992) Nat, Prod. Rep. 9: 323; Faulkner (1993) Nat, Prod. Rep. 10: 497; Faulkner (1994) Nat, Prod. Rep. 11: 355; Faulkner (1954) Nat, Prod. Rep. 12: 223; Faulkner (1996) Nat, Prod. Rep. 13: 75; Faulkner (1997) Nat, Prod. Rep. 14: 256; and Faulkner (1985) J. Am. Chem. Soc. 107: 4796-4798). However there exist literally thousands of species of marine sponges and these organisms are only beginning to be explored.
This invention provides novel compounds derived from a marine sponge, Haliclona (aka Adocia) sp., that specifically modulate (e.g., inhibit) kinesin activity by targeting the kinesin motor domain and mimicking the activity a microtubule. It is believed this mode of kinesin motor modulation is previously unknown. Thus, it was also a discovery of this invention that the kinesin-microtubule interaction site is a useful target for small molecule modulators of kinesin motor activity.
Because the compounds were initially derived from the marine sponge Haliclona (Adocia) sp. they are referred to herein as Adocia compounds or Adocia-derived compounds. Particularly preferred Adocia-derived compounds are adociasulfates. Thus, in one embodiment, this invention provides for the Adocia-derived compounds having the formulas shown herein, more preferably for the adociasulfate compounds having the formulas shown herein.
The Adocia-derived compounds are potent kinesin motor modulators that appear to block kinesin binding of microtubules. The compounds are potent anti-mitotic agents that are highly effective in vitro and in vivo. Thus, in another embodiment, this invention provides composition for the in vivo modulation (e.g., inhibition) of kinesin motor activity (e.g., in a cell). The compositions typically comprise any of the Adocia-derived kinesin motor inhibitors described herein in combination with a pharmacologically acceptable excipient.
In another embodiment, this invention provides methods of modulating (e.g., inhibiting) kinesin motor activity in a cell. The methods involve contacting the cell with one or more of the Adocia-derived kinesin modulators described herein. The cell, although preferably a mammalian cell, need not be so limited. Other suitable cells include, but are not limited to, fungal cells and microbial cells. The cell can be in vitro or in vivo. Where the method is practiced in a therapeutic context (e.g., to ameliorate the effects of a pathological condition characterized by hyperproliferation of one or more cells) the Adocia-derived kinesin modulators are preferably administered in a therapeutically effective dose.
In still another embodiment, this invention provides methods of assaying a test compound for kinesin modulatory activity. The methods involve contacting a microtubule and a kinesin motor (e.g. a kinesin motor protein) with one of the Adocia-derived kinesin modulators described herein and detecting a change in kinesin motor activity resulting from the contacting. In a particularly preferred embodiment, the method is practiced with one of the kinesin modulators of Formulas I, and III-VI, more preferably with one of the kinesin modulators of Formulas I or III. The change in motor activity is preferably detected through a motility assay, a binding assay, an ADP release assay, or an assay for anti-mitotic activity. Typically the change in activity is evaluated with reference to a negative control (e.g., typically the same assay, but lacking a kinesin motor modulator) and/or with reference to a positive control (e.g., typically the same assay, with a different kinesin motor inhibitor, preferably one whose activity has previously been characterized).
This invention also provides Adocia-derived kinesin modulator kits. The kits typically include a container containing one or more of the Adocia-derived kinesin modulator described herein. The kits can optionally include a pharmacological excipient and/or a delivery vehicle. When the excipient and/or delivery vehicle are provided they may be provided combined with the kinesin motor inhibitor or in a separate container for combination at the time of use. The kit can also include instructional materials describing the use of the compounds in any of the methods described herein.
In still another embodiment, this invention provides methods of modulating kinesin motor activity. The methods involve contacting the kinesin motor with a small organic molecule that competitively inhibits the kinesin motor at a microtubule binding site. in a particularly preferred embodiment, the small organic molecule is an Adocia-derived kinesin modulator as described herein or a a small organic molecule is identified according to the methods described herein.
This invention also provides methods of identifying an agent that modulates the kinesin inhibitory activity of an Adocia kinesin inhibitor (e.g., on of the Adocia sulfates or Adocia derived kinesin inhibitors described herein). The methods involve contacting a microtubule and/or a kinesin motor and/or an Adocia kinesin inhibitor with a candidate agent; and detecting a change in the kinesin inhibitory activity of the Adocia kinesin inhibitor resulting from the contacting, wherein a change indicates the identification of an agent that modulates the kinesin inhibitory activity of the Adocia kinesin inhibitor.
Methods are also provided for identifying an agent that interferes with the binding of an Adocia kinesin inhibitor with a kinesin. These methods involve contacting a kinesin and an Adocia kinesin inhibitor (e.g. an adocia sulfate or an adocia derived kinesin modulator described herein) with a candidate agent; and detecting a decrease in the binding of the Adocia kinesin inhibitor with the kinesin resulting from said contacting, wherein a decrease indicates the identification of an agent that interferes with the binding of the Adocia kinesin inhibitor and the kinesin.
Also provided herein is a complex comprising an Adocia kinesin inhibitor and a kinesin.
Methods are also provided for modulating cellular growth in an organism (e.g., an animal or a plant). The methods preferably involve administering to the organism a composition comprising a pharmaceutically acceptable carrier any one or more of the compounds described herein (e.g. adocia sulfates or adocia-derived kinesin modulators) in a quantity sufficient to alter said cellular growth in an organism.
Definitions
The term xe2x80x9cmolecular motorxe2x80x9d refers to cytoskeletal molecule(s) that utilize chemical energy to produce mechanical force, and drive the motile properties of the cytoskeleton.
The terms xe2x80x9ckinesinxe2x80x9d and xe2x80x9ckinesin superfamilyxe2x80x9d as used herein refer to a superfamily of eucaryotic motor proteins used to transport a large variety of cargoes along microtubule xe2x80x9ctracksxe2x80x9d. Members of the kinesin superfamily are believed to be essential for mitotic and meiotic spindle organization, chromosome segregation, organelle and vesicle transport and many other processes that require microtubule based transport. The common feature of kinesins in the presence of a conserved xcx9c350 amino acid motor domain which harbors the microtubule binding, ATP-hydrolyzing, and force transducing activities (see, e.g., Barton et al. (1996) Proc. Natl. Acad. Sci. USA, 93(5): 1735-1742, and Goldstein, (1993) Annu. Rev. Genet., 27: 319-351).
The term xe2x80x9ckinesin motorxe2x80x9d is used to refer to one or more proteins involved in the transduction of chemical energy into mechanical energy. Kinesin is a force generating enzyme that hydrolyzes ATP to ADP and Pi and uses the derived chemical energy to induce plus end directed movement along microtubules. This ubiquitous microtubule motor is thought to power anterograde organelle transport along microtubules. The term kinesin motor is intended to include kinesin related proteins inhibition of which inhibits kinesin motor activity. Kinesin heavy and light chains have been cloned and sequenced from a number of species including, but not limited to Drosophila (GenBank M24441), squid optic lobe (GenBank J05258), sea urchin and human (GenBank X65873), and rat (M75146, M75147, M75148), and the like (see, e.g., Yang et al. (1989) Cell 56: 879-889, Wright et al. (1991) J. Cell. Biol., 113: 817-833, Navone et al. (1992) J. Cell. Biol., 117: 1263-1275, and Cyr et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 10114-10118). In addition, the scientific literature is replete with detailed descriptions of kinesins (kinesin motors) and kinesin related proteins (see, e.g., Kreis and Vale (1993) Guidebook to the Cytoskeletal and Motor Proteins, Oxford University Press, Oxford, Vale (1990) Curr. Opin. Cell. Biol. 2: 15-22; Vale (1987) Ann. rev. Cell. Biol., 3: 347-378; and references therein).
The terms xe2x80x9ckinesin motor inhibitorxe2x80x9d or xe2x80x9cinhibition of kinesin motor activityxe2x80x9d refers to the decrease or elimination of kinesin/microtubule mediated transduction of chemical energy (e.g. as stored in ATP) into mechanical energy (e.g., force generation or movement). Such a decrease can be measured directly, e.g., as in a motility assay, or alternatively can be ascertained by the use of surrogate markers such as a decrease in the ATPase activity of the kinesin protein, and/or a decrease in the affinity and/or specificity of kinesin motor protein-microtubule binding interactions, and/or in a decrease in mitotic activity of a cell or cells. Conversely, a xe2x80x9ckinesin motor agonistxe2x80x9d or xe2x80x9cupregulator of kinesin motor activityxe2x80x9d refers to the increase of kinesin/microtubule mediated transduction of chemical energy (e.g. as stored in ATP) into mechanical energy (e.g., force generation or movement).
An xe2x80x9cAdocia-derived compoundxe2x80x9d or xe2x80x9cAdocia-derived kinesin modulatorxe2x80x9d as used herein refers to any of the kinesin modulators described herein (see, e.g. Formulas I, III, IV, V, and VI). It will be appreciated, that while the Adocia-derived compounds include natural products derived from sponges (or other marine organisms) the term also contemplates analogues of such compounds as described herein. The Adocia-derived compounds thus need not exist as natural products and may be chemically synthesized de novo.
The term xe2x80x9ctest compoundxe2x80x9d refers to a compound whose anti-kinesin motor activity it is desired to determine. Such test compounds may include virtually any molecule or mixture of molecules, alone or in a suitable carrier.
The term xe2x80x9cdetecting the bindingxe2x80x9d means assessing the amount of a given second component that binds to a given first component in the presence and absence of a test composition. This process generally involves the ability to assess the amount of the second component associated with a known fixed amount of the first component at selected intervals after contacting the first and second components. This may be accomplished e.g., by attaching to the second component a molecule or functional group that can be visualized or measured (e.g., a fluorescent moiety, a radioactive atom, a biotin that can be detected using labeled avidin) or by using ligands that specifically bind to the second component. The level of binding is preferably detected quantitatively. Binding, or a change in binding is indicated at the first detectable level. A change in binding, which can be an increase or a decrease, or presence versus absence, is preferably a change of at least about 10%, more preferably by at least about 20%, still more preferably by at least about 50%, still even more preferably by at least about 75%, even more preferably by at least about 150% or 200% and most preferably is a change of at least about 2 to about 10 fold (e.g., as compared to a control).
The phrase xe2x80x9cdetecting a change in the kinesin inhibitory activity of he Adocia kinesin inhibitor resulting from said contactingxe2x80x9d refers to determining the presence or absence or quantifying the alteration in kinesin inhibitory activity caused by a particular candidate agent, assays for such determinations are further described herein. A change in activity, which can be in increase or a decrease, or presence versus absence, is preferably a change of at least about 10%, more preferably by at least about 20%, still more preferably by at least about 50%, still even more preferably by at least about 75%, even more preferably by at least about 150% or 200% and most preferably is a change of at least about 2 to about 10 fold (e.g., as compared to a control).
The phrase xe2x80x9cdetecting a change in kinesin motor activity resulting from said contactingxe2x80x9d refers to determining the presence, absence or quantifying the alteration in kinesin motor activity caused by a particular composition (e.g., a test compound). The detecting can involve any one or more of a variety of assays for kinesin motor activity as described herein. A change in activity, which can be an increase or a decrease, or presence versus absence, is preferably a change of at least about 10%, more preferably by at least about 20%, still more preferably by at least about 50%, still even more preferably by at least about 75%, even more preferably by at least about 150% or 200% and most preferably is a change of at least about 2 to about 10 fold (e.g., as compared to a control).
The term xe2x80x9ccompoundxe2x80x9d as used herein refers to organic or inorganic molecules. The term includes, but is not limited to polypeptides, proteins, glycoproteins (e.g. antibodies), nucleic acids, oligonucleotides, and inorganic molecules.
The term xe2x80x9csmall organic moleculexe2x80x9d, as used herein, refers to a compound that is an organic molecules of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
A xe2x80x9cbioagricultural compoundxe2x80x9d as used herein refers to a chemical or to a biological compound that has utility in agriculture or in environmental and functions to foster food or fiber crop or crop protection or yield improvement. For example, one such compound may serve as a herbicide to selectively control weeds, as a fungicide to control the spreading of plant diseases, as n insecticide to ward off and/or destroy insect, mite, and other arthropod pests. In addition, one such compound may demonstrate utility in seed treatment to improve the growth environment of a germinating seed, seedling, ro young plant as a plant regulator or activator. Other compounds can serve in environmental management such as, for example, forest management.
By xe2x80x9cproteinxe2x80x9d herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides, and peptides. The protein may be made of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus xe2x80x9camino acidxe2x80x9d, or xe2x80x9cpeptide residuexe2x80x9d, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline, and norleucine are considered amino acids for the purposes of this invention. xe2x80x9cAmino acidxe2x80x9d also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
The terms xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9coligonucleotidexe2x80x9d or grammatical equivalents herein refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 141 9), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321, O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside and Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, xe2x80x9cCarbohydrate Modifications in Antisense Researchxe2x80x9d, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic and Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp169-176). Several nucleic acid analogs are described in Rawls, C and E News Jun. 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
The term xe2x80x9ccompetitive inhibitionxe2x80x9d is used to refer to competitive inhibition in accord with the Michaelis-Menton model of enzyme kinetics. Competitive inhibition is recognized experimentally because the percent inhibition at a fixed inhibitor concentration is decreased by increasing the substrate concentration. At sufficiently high substrate concentration, Vmax can essentially be restored even in the presence of the inhibitor. Conversely, xe2x80x9cnon-competitive inhibitionxe2x80x9d refers to inhibition that is not reversed by increasing the substrate concentration.
The term xe2x80x9ccellxe2x80x9d is used to refer to any cell including, but not limited to mammalian, fungal, microbial and invertebrate cells. Preferred cells include tumor cells including, but not limited to, carcinomas, including breast, ovary, prostate, skin, and colon; brain cancers, including memingioma, glioma, oligodendroglioma, embryonic cancers; sarcomass; leukemias, and lymphomas. Preferred cells also include neurons. Particularly preferred neurons are those related to neurodegenerative diseases including Alzheimer""s Disease, Parkinson""s Disease, Huntington""s Disease, Frontotemporal Dementias, and Amyotrophic Lateral Sclerosis. Preferred cells further include cells derived from the gastrointestinal system including esophagus, stomach, intestine, pancreas, liver, lung, heart, and vascular system as sell as cells from the central and peripheral nervous system, kidney, bladder, muscular system and the bone system.
xe2x80x9cIn vivoxe2x80x9d refers to in the living body of an organism.
xe2x80x9cIn vitroxe2x80x9d refers to outside the living body, such as, an artificial environment, for example, a test tube or a cell or tissue culture.
The term xe2x80x9cmodulatexe2x80x9d as used herein refers to increaing or decreasing an activity of a molecule. Thus, for example, a kinesin motor modulator acts to increase or decrease (inhibit) kinesin motor activity.